Lumpy skin disease (LSD) is a transboundary viral disease of cattle that causes substantial economic loss globally. There is no specific treatment and subunit vaccine for this disease to date. Reports of the global spread of this disease are worrisome. We designed a multi-epitope protein using an immunoinformatics approach in this study. We analyzed the proteome of LSDV and found 32 structural/surface proteins. Four of these 32 proteins were predicted as antigenic and non-homologous to bovine and highly conserved in 26 LSDV isolates. The predicted B-cell epitopes and CTL epitopes were stitched together with the help of an AAY linker leading to the formation of a multi-epitope protein. The in silico study revealed that the modeled subunit vaccine candidate and TLR4 receptor interact with high affinity. This interaction was also found to be stable using a molecular dynamics simulation study. Our study demonstrates a leap towards developing a subunit vaccine against LSD. Poxviridae and genus Capripoxvirus. The LSDV is a double-stranded DNA virus. The genome size of LSDV is 151 kb. The genome consists of 156 putative genes1 of which 146 genes are conserved that encode proteins involved in transcription, mRNA biogenesis, virion structure, and assembly1. The genes of LSDV are akin to the genes of other viruses of the Capripoxvirus genus, e.g., sheeppox virus (SPPV) and goatpox virus (GTPV)2. LSDV has a limited host range. It only causes infection in cattle (Bos indicus and Bos taurus) and buffaloes 3. LSD leads to high morbidity and low mortality in infected animals. The high morbidity caused by LSD significantly lowers the productivity of animals4. The clinical signs of the disease include fever, enlarged lymph nodes, drop in milk production, anorexia, abortion, infertility, and characteristic nodules on the skin4. The clinical symptoms of LSD was first described in Zambia in 19295. The present geographical distribution of this disease is spread over in Austalian, African, European and Asian countries7. Acknowledging the economic impact of LSD, the World Organization of Animal Health (OIE) categorized LSD as a notifiable disease8. Also, due to the ability of this virus to spread rapidly, it is considered an agro-terrorism agent9.Lumpy skin disease (LSD) is a transboundary viral disease of cattle caused by the lumpy skin disease virus (LSDV). LSDV is an enveloped virus belonging to the family 12. Assays are also designed to differentiate between the wild-type and the vaccine strains14. Serology is based on neutralization tests but cannot discriminate antibodies (raised by infection/immunization) or virus species 17.Clinical signs such as skin nodules observed on the face, neck, udder, limbs etc., are used to diagnose LSD. The laboratory diagnosis of LSDV is primarily based on qPCR approaches that are mostly genus-specific4. Also, live attenuated LSDV vaccine strains like Neethling have been developed4. However, live attenuated/inactivated vaccines are widely used and considered effective for veterinary usage, but they have limitations. Furthermore, the reports of recombination of LSDV vaccine strains and field strains19 are worrisome. Also, there are chances of revival of live attenuated virus in the immunocompromised animals. These issues with live attenuated virus strategies for LSDV compelled us to look for an alternative like a subunit vaccine proactively. The subunit/multi-epitope-based vaccines are generally considered safer and cost-effective compared to the live attenuated vaccines20. Hence development of a subunit/multi-epitope vaccine for LSD is highly desirable21.No chemotherapeutic drugs are available for the treatment of LSD and there are no subunit vaccines for the control of LSD. Current control of this disease is possible through vaccination with the live attenuated SPPV vaccine but is restricted to those countries where there is an overlap between SPPV, GTPV and LSDV infection23. The exploitation of the immunoinformatics approach before experimental analysis is advantageous as it saves both time and money in addition to the experimental efforts. Furthermore, it can also narrow down a vast number of targets to a few potential molecules. Numerous studies have shown that multi-epitope vaccines could effectively elucidate protective immunity against influenza A, hepatitis B, and hepatitis C viruses26. Researchers have designed multi-epitope vaccine candidates for zoonotic viruses such as Nipah Virus, Crimean Congo Hemorrhagic Fever virus, Marburg virus, Ebola virus, and Monkeypox virus31. Also, few studies have shown that subunit vaccines against foot and mouth disease virus can be effective for veterinary applications34.Immunoinformatics integrates computational and molecular immunological tools for identifying target antigens for vaccine developmentWe used an immunoinformatics-driven approach in this study to design a subunit vaccine candidate against LSDV that could elicit a protective humoral and cellular immune response. Here, we designed a novel multi-epitope protein from LSDV in such a way that it displays various antigenic epitopes from LSDV.A systematic workflow was designed using the immunoinformatics approach to identify the potential vaccine candidates from the proteome of the LSDV Fig. . We subs1 . We found that a protein with the gene-ID LSDV123 was homologous to the bovine c type lectin domain family 7 member A isoform X2 (ID-XP_005207118.1), whereas the other 4 proteins with gene-IDs as LSDV031, LSDV090, LSDV103, and LSDV109 were non-homologous to bovine  only with 22 different bovine MHC-I molecules  epitopes using NetCTL 1.2 server against all the 12 supertypes. We selected the threshold of 0.98 for better sensitivity and specificity in predicting the CTL epitopes. Seventy-seven CTL epitopes were identified from the NetCTL server. The CTLs recognize antigens in the context of MHC-I. Hence, estimating the binding affinity of the CTL epitope and bovine MHC-I molecule is essential. All the 77 CTL epitopes were further analyzed for their binding affinity with a total of 77 bovine MHC-I molecules of The crystal structures for the selected bovine MHC-I, namely BoLA-1*00902, BoLA-3*05101, BoLA-6*01301, BoLA-5*00301, BoLA-6*01402, and BoLA-2*01201 proteins, were unavailable. Hence a homology model-based approach was carried out to build a reliable model structure for these selected proteins using Modeller 9.19. Only the extracellular region of these proteins was considered for modeling . After refinement, the RMSD value of the modeled BoLA-6*01301 and BoLA-6*01402 were predicted to be 1.084 Å and 0.687 Å, respectively. The Ramachandran plot of all the modeled bovine MHC-1 suggested that the predicted models were stable and reliable  was found to have 71.88% identity and 72% coverage with the bovine TLR4. Similarly, the human TLR2 (PDB Id: 6ING_A) was found to have 68.37% identity and 69% coverage with the bovine TLR2. A reliable model for bovine TLR2 and TLR4 was predicted using Modeller 9.19 Figs. A and 3A.The linear amino acid sequence of multi-epitope protein against LSDV was further analyzed for the presence of B-cell and CTL epitopes using BCPREDS and NetCTL, respectively. From BCPREDS analysis we found that the multi-epitope protein contains a total of 14 B-cell epitopes and all the B-cell epitopes were identical to the previously predicted epitopes. Similarly, we found from the NetCTL and HLArestrictor analysis that the multi-epitope protein against LSDV possesses a total of 30 CTL epitopes. Of these 30 CTL epitopes, 17 were previously identified as CTL cell epitopes, and 13 newly predicted CTL epitopes were identified. All the 13 newly predicted CTL epitopes were further verified for their homology with bovine, and we found none of these 13 epitopes were present in bovine  were calculated using the PatchDock server from the refined modeled structure of both proteins. Twenty protein–protein complexes were generated based on the protein surface, geometry and electrostatic complementarity. Top 10 of these 20 protein–protein complexes were again submitted to the FireDock to re-score the complexes according to their global binding energies. The final docking model for TLR2 and multi-epitope protein was found to be 6.5 kcal/mol (least binding energy score) this suggests that TLR2 would not interact with the multi-epitope protein Fig. B. Hence,ly Figs. . The resly Figs. . This reWe used immunoinformatic tools for the prediction of the potential antigens of LSDV, which are (a) predicted to be on the surface so that it can be targeted by the host immune response, and (b) non-homologous to the host protein. We selected 32 structural/surface proteins from the LSDV proteome to identify the immunogenic proteins. All these proteins were analyzed in silico for their ability to induce an immune response. We used Vaxijen server for the prediction of potential antigens. Vaxijen is an antigen prediction server based on an alignment-free approach using autocovariance (ACC) transformation of protein sequence into uniform vectors of principal amino acid properties. Further, it is critical that a vaccine candidate should not generate autoimmunity in the host. Hence, these selected proteins were found to be non-homologous with the host proteome. The genetic diversity analysis among selected proteins showed that these proteins are conserved in various isolates of LSDV.38. Hence, we identified B-cell and CTL-epitopes while designing the multi-epitope protein in silico. However, due to limited resources/knowledge available to study bovine immune system, we could not perform analysis of bovine HTL epitopes. For considering HTL epitopes, we need to screen the bovine MHC-II molecules; however, to date, there are no resources/software(s) available which can be used to screen peptide sequences against the bovine MHC-II molecule.B-cells play an essential role in the development of humoral immunity. Although cell-mediated immunity plays a vital role in the clearance of the virus, the role of B-cells in enhancing cell-mediated immunity cannot be ignored. During viral infection, B cells function as antigen-presenting cells by presenting viral peptides to CD4 + cells through MHC-II39. Although LSDV does not infect DC, cross-presentation of viral antigen may be possible. Hence, we predicted CTL epitopes and their corresponding MHC-I molecule in the selected LSDV proteins. We observed that BoLA-1*00902, BoLA-3*05101, BoLA-6*01301, BoLA-5*00301, BoLA-6*01402, and BoLA-2*, were sufficient to bind to all the selected CTL epitopes. Hence, we confined our studies to only these MHCs. The modeled MHC-I and CTL-epitopes were docked with the help of PyRx software, and their binding energies were calculated further to validate the interaction of modeled MHC-I and CTL-epitopes.Generally, cell-mediated immunity plays an important role in viral clearance. It has been shown that the viruses which infect dendritic cells (DC) can directly present viral antigens to CTL. However, the viruses that target other cells different from the DC cannot directly present viral antigens to CTL. In this case, cross-presentation of viral antigens by DC to activate virus-specific CTL is required40. This study suggested that the amino acids with aromatic (tyrosine), basic (lysine), and small aliphatic side chains  support efficient CTL recognition of both flanking epitopes. Hence, we used an AAY linker for joining the predicted CTL epitopes and B-cell epitopes. In order to understand the effect of AAY linker on the immunogenicity and stability of multi-epitope vaccine candidate, we designed the multi-epitope vaccine candidate with and without a linker. The antigenicity of the multi-epitope vaccine candidate with and without linker was found to be 0.5791 and 0.5158, respectively. This suggests that adding the linker AAY would not affect the immunogenicity of the vaccine candidate. The physiological properties of the vaccine candidate was also predicted with and without AAY linker. The instability index of the multi-epitope protein without AAY linker was found to be 41.75, suggesting that the protein would be unstable. In contrast, after adding the linker AAY the instability index of the multi-epitope protein was found to be 37.95, suggesting the protein would be stable. Further, we used the FoldIndex server to predict the unfoldability index of the multi-epitope vaccine candidate with and without a linker. We observed that the multi-epitope vaccine candidate with and without linker AAY linker would have a similar unfoldibilty index  protein leading to the induction of IL-644. Recently, various other viral glycoproteins, such as Ebola virus GP45, VSV-G46, the envelope proteins of Moloney murine leukemia virus (MMLV)47, could bind and activate TLR4, further leading to the expression of proinflammatory cytokine expression. Thus we analyzed the potential of TLR4 receptor to interact with the modeled multi-epitope vaccine candidate, in silico, in this study. An effective CTL response against the designed multi-epitope protein would be generated only when it is presented to the CD8 + T-cells by the antigen-presenting cells. Thus, we hypothesized that bovine TLR4 present on the antigen-presenting cells would play an important role in recognizing the multi-epitope protein. The subunit vaccine candidate was found to interact with TLR4. Further, the docking and molecular dynamics simulation studies of the bovine TLR4 and the multi-epitope protein suggested that this complex would be stable. The interaction of TLR4 with subunit vaccine candidate would activate TLR4. The activation of TLR4 pathway would activate cytokines further, leading to the activation of both innate and adaptive immune systems. Thus, in this study, we designed a new multi-epitope protein of LSDV, which would likely be an effective subunit vaccine candidate against LSDV.Toll-like receptors (TLRs) are essential in recognizing conserved molecules derived from microbes. They are characterized by an extracellular leucine-rich repeat domain and an intracellular Toll/IL-1 receptor-like (TIR) domain. Of all the TLRs, only TLR4 and TLR2 have been shown to recognize the viral proteinsIn conclusion, we have designed a multi-epitope protein in silico from the structural proteins of LSDV using an immunoinformatic approach. The engineered subunit vaccine protein has been found to interact with the toll-like receptor and found to be stable under in silico conditions. Although the subunit vaccines have various advantages, they may have their own limitations. One of the significant limitations is the expression and purification of the recombinant protein as a soluble protein in heterologous expression systems. It will be interesting to confirm the expression and purification of the designed multi-epitope subunit vaccine candidate. Also, the validation of predicted immunogenicity and the potential of this designed multi-epitope protein to interact with the TLR4 in vivo would require for subsequent trials in the animals. In short, our studies demonstrate a way toward developing a subunit vaccine against LSDV and put forward a new multi-epitope protein as a potential subunit vaccine candidate likely to be effective against LSDV.1 were downloaded from the National Centre for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov) for the antigenicity prediction. The antigenicity of the identified surface/structural proteins was analyzed using Vaxijen server35. The threshold was set to 0.5 to predict the antigenicity of proteins. Further, to identify the non-homologous proteins of Bos taurus with LSDV surface proteins, the BLASTp server48 was used at a threshold of E-value at 0.005.The surface/structural proteins of the lumpy skin disease virus NI-2490 isolate Neethling 2490 (Accession no. AF325528.1) reported previously49.The proteomes of 27 isolates of LSDV were downloaded from the NCBI database. The selected proteins were searched against the proteome of these 27 isolates of LSDV and were aligned together using Clustal OmegaBos taurus and predicted to be antigenic) from the previous step were analyzed for the prediction of the B-cell epitope(s) using BCPREDS with a specificity of 75% and a peptide score of > 950. BCPREDS training dataset contains 701 linear B-cell epitopes, and 701 random B-cell epitopes from Bcipep and SwissProt sequence database, respectively. The transmembrane region was predicted for all the selected proteins using TMHMM Server, v. 2.051.The selected proteins (non-homologous with 52 was used to predict the presence of cytotoxic T-lymphocyte (CTL) epitopes against all the 12 supertypes with a threshold of 0.98. The CTL epitopes identified from the NetCTL 1.2 were further screened for the identification of bovine MHC-I restriction element using HLArestrictor 1.153 against all the 77 bovine MHC-I alleles present in the database. The strong binders were classified with a threshold of 50 for IC50 and 0.5 percentile rank, while the weak binders were classified with a threshold of 500 for IC50 and 2 percentile rank.NetCTL 1.2 server54. The transmembrane regions for all the bovine MHC-I molecules were predicted by TMHMM Server, v. 2.051. Only the extracellular region of MHC-I was used to build a reliable tertiary structure for these proteins using a homology model-based approach. The template for the homology modeling was identified by a BLASTp search against Protein Data Bank (PDB) and the three-dimensional models of selected BoLA-MHC-I proteins were built using the MODELLER 9.19 software55. The stereochemistry and conformation analysis of generated models for these proteins were analyzed using PROCHECK, a protein parameters analysis tool56 and refinement was done using ModRefiner57.The sequences of bovine MHC-I alleles, namely BoLA-1*00902, BoLA-3*05101, BoLA-6*01301, BoLA-5*00301, BoLA-6*01402 and BoLA-2*01201, were downloaded from the Immuno Polymorphism Database (IPD)58 was used for the prediction of the three-dimensional structure of CTL epitopes (classified as a strong binder from the HLArestrictor 1.1). The parameter was set to generate 5 probable structures of the CTL epitopes. The structure with the lowest energies was selected as the final model for the respective epitope.The PEP-FOLD server59 was used for docking studies between the strong binder pairs of MHC-I allele and CTL epitope obtained from HLA-restrictors. For BoLA-1*00902, BoLA-3*05101, BoLA-2*01201, and BoLA-5*00301, the grid was set to 106.281, 107.904, 86.508 for center X, Y, and Z coordinates, respectively, with a spacing dimension of 110.355 Å, 102.153 Å, and 113.218 Å for the X, Y, and Z coordinates, respectively. In the case of BoLA-6*01402 and BoLA-6*01301, the grid box was set to 76.564, 57.040, 23.777 for X, Y, and Z coordinates, respectively with the spacing dimension of 72.848 Å, 81.183 Å, 88.263 Å for the X, Y, and Z coordinates, respectively. The binding affinity of the CTL epitope with its receptor BoLA-MHC-I protein was measured in kcal/mol.The PyRx software60 and FoldIndex server61, respectively. Also, ProtParam62 was used to predict the physicochemical properties, such as amino acid composition, theoretical pI, instability index etc., of the designed subunit vaccine candidate.A subunit vaccine candidate for LSDV was designed by fusing all the CTL and B-cell epitopes with an AAY linker. A few extra amino acids that were in continuation with the epitope of the same protein were added to maintain the integrity of the epitopes and to prevent the generation of newer epitopes after joining with the linker. The presence of homologous sequences with bovine in the designed subunit vaccine candidate was done using BLASTp. The antigenicity, allergenicity, and intrinsically unfolded regions were predicted using Vaxijen, AlgPred63, is a distance-based protein folding method of structure prediction, was used for tertiary structure prediction of the designed subunit vaccine candidate. The reliable models for bovine TLR4 and TLR2 were built using homology modeling. A BLASTp search against PDB database was used to identify the best template for homology modeling for bovine TLR4 and TLR2 proteins. The three-dimensional model of bovine TLR4 and TLR2 proteins were built using MODELLER 9.19 software. The model refinement and stereochemistry analysis for the subunit vaccine candidate and above modeled bovine TLR (TLR4 and TLR2) were achieved using the ModRefiner and PROCHECK, respectively.The RaptorX server64, was used to study the interaction between the bovine TLRs (TLR4 and TLR2) and the subunit vaccine candidate. The top ten models were again submitted to FireDock server65 for sorting them according to their global binding energies. To further validate the interaction of TLR4 and multi-epitope protein, we used ClusPro server37. Further, molecular dynamics (MD) simulation study was carried out for the docked complex of the TLR4 and subunit vaccine candidate for 100 ns with a time interval of 2 fs using Gromacs software to determine the stability of the protein–protein complex. The simulation was done using Optimized Potentials for Liquid Simulations (OPLS) force filled parameters. The receptor-multi-epitope protein complex was solvated with SPCE water molecules. The isothermal-isobaric pressure (NPT) ensemble was set at 300 K (temperature) and 1 atm pressure. The root mean square deviation (RMSD) was calculated to examine the standard deviation of the backbone and sidechain of the protein. Further, to calculate the binding free energy of the complex MMGBSA analysis performed at the HawkDock server66.The protein–protein docking server, PatchDockSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions , passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS–PAGE. The secondary structure of SACMV MP presented as predominantly β-strands. An ANS -binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS . A 2′ (3′)-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells  Geminiviridae and genus Begomovirus, whose members infect a wide range of plant hosts  (mdeg cm2 dmol−1) using the following formula:c is the concentration in g/L, n is the number of residues, and l is the path length in cm.Far-UV circular dichroism (Far-UV CD) measurements of MP were performed at 20 °C in a Jasco J1500 spectrometer. Briefly, the protein sample was diluted to a final concentration of 5 µM using ultra-pure deionised water, which resulted in a final Tris buffer containing 0.5 mM Tris-HCl (pH 7.5). Three independent spectra were collected for both the protein sample  and the buffer (as blank). All spectra were corrected for using the Tris-HCl buffer as a baseline. The raw data were converted to mean residue ellipticity [The estimation of secondary structure of BC1 was estimated using the CONTINL algorithm in the Dichroweb online server .3, and stored in the dark. The concentration of ANS was determined using UV–Vis absorbance at 350 nm. All measurements were performed in a 50 mM Tris-HCl (pH 10) and 0.5 M l-arginine buffer. Each protein sample (triplicate) was equilibrated in the buffer, and 10 µM protein was used for each measurement. In order to determine what occurs in denatured proteins, measurements were also performed in 8 M urea  in 50 mM Tris-HCl (pH 8.5) and 0.05% (w/v) NaN3 (pH 7–7.5). Each sample was excited at 380 nm, and three emission spectra between 400 and 600 nm were collected and averaged.The extrinsic ANS fluorescence spectra for the SACMV MP were measured with a Jasco FP-6300 spectrofluorometer. A 20 mM stock of ANS was resuspended in 100 mM sodium phosphate (pH 6.5), 1 mM EDTA, and 0.02% NaNl-arginine) and in 8 M urea . Per 100 µM of mant-ATP, 35 µM of protein was used (ratio 20:7). Each sample was excited at 360 nm, and three emission spectra between 400 and 600 nm were captured and averaged.The ability of the hydrophobic sites of the MP to bind mant-ATP was determined using fluorescence spectroscopy. The fluorescence spectra were measured using a Jasco FP-6300 spectrofluorometer. A 100 µM stock of an original 5 mM stock of mant-ATP  was resuspended in refolding buffer  was used to determine the amount of free phosphate released. Experiments were prepared and measured as per the manufacturer’s instructions. All measurements were recorded at 620 nm on a Multiskan spectrophotometric plate reader. All background absorbance was corrected by subtracting background values from the standard phosphate absorbance.l-arginine or 8 M urea. Background fluorescence (free unbound tryptophan residues) was corrected by subtracting background values from the actual protein spectra (protein bound to tryptophan residues).Intrinsic tryptophan fluorescence was carried out in order to determine if the protein was refolded and if there were any conformational changes in the presence and absence of ATP. Measurements were performed in the presence and absence of ATP and for the native and denatured forms of the protein. Six samples were analysed using a Jasco FP-6300 spectrofluorometer. Tryptophan residues were excited at 280 nm, and fluorescence spectra were recorded between 300 and 500 nm. Each spectrum is the average of three accumulation replicates in 50 mM Tris-HCl (pH 7.5), containing 0.5 mM The optimal expression conditions for this protein were successfully obtained in this study by varying induction temperature , IPTG concentration , and induction period (0–24 h). Based on the results, it was demonstrated that SACMV MP is predominantly insoluble A,B and iAffinity chromatography is a technique commonly selected for the purification of proteins. A number of His-tagged proteins are often purified by means of affinity chromatography . For thiFar-UV CD spectroscopy (between 180 and 250 nm) estimates the secondary structure content of proteins and peptides and determines the fraction of α-helices and β-strands with respect to the amino acids involved in unordered forms in an amide backbone . The farmax) from 510 nm to 480 nm for MP (native form). This indicates that MP provides ANS a non-polar environment upon binding, suggesting that MP possesses hydrophobic binding pockets in its native form. The denatured MP showed a significant shift in quantum yield but did not show a blue shift (λmax) from 510 nm to 480 nm (λmax remained at 510 nm) (The tertiary structure of MP was examined with the aim of identifying possible hydrophobic sites in the protein.  510 nm) . This inmax) in the spectra between the buffer and the native protein. In the denatured form (presence of 8 M urea), SACMV MP was unable to bind the mant-ATP.The ATP binding assay uses mant-ATP, in which the ATP molecule displays fluorescence at 360 nm and reveals the degree to which a protein binds ATP ,14. WhenThe phosphatase assay was used to determine whether SACMV MP was able to hydrolyse ATP to form ADP and Pi or AMP and PPi . Resultsmax) from 375 nm to 345 nm for native MP in the absence of ATP and native MP in the presence of ATP even though there is a significant drop in quantum yield, compared with the buffer control. Tryptophan fluorescence in this study was utilised for the purpose of monitoring the conformational changes in MP and is also used to monitor the successful refolding of proteins. SACMV MP contained three tryptophan residues. E. coli host chosen. Bacteria such as E. coli, while preferred due to their fast growth rate, are also known for possible misfolding and precipitation of heterologous proteins [Pichia pastoris.While low temperatures usually favour soluble protein expression ,17,18, tproteins ,22. An aproteins . While iArabidopsis thaliana [Intrinsically disordered proteins or proteins with intrinsically disordered regions at the N- or C-termini often present difficulties with protein expression, purification, and crystallisation. Therefore, it is advised that disordered regions of a protein of interest are predicted before any structural studies are pursued . Based othaliana . The intSince structural studies on geminiviral MPs are absent from the literature, it is not known if ATP or other biomolecules bind to these proteins to form a complex in order to facilitate movement in the cell. In some geminiviruses, such as the Squash leaf curl virus (SqLCV), the MP traps the NSP–ssDNA complexes in the cytoplasm to move them to adjacent cells , suggestmax)  . This suArabidopsis homologs HSP90 (AT5G56030) and mitochondrial HSP60 (AT3G23990) were demonstrated to interact with SACMV MP via Y2H and co-IP in cassava infected with SACMV. Since the MP has the proposed ability to bind ATP, further kinetic research may be carried out in future studies. In addition, further structural and molecular characterisation studies of geminivirus cell-to-cell movement proteins will unravel more in planta features of their functions.In conclusion, this research on the SACMV MP revealed, for the first time, the proposed secondary structure of a denatured refolded full-length monomeric geminivirus cell-to-cell movement protein in vitro. Due to the lack of attainment of soluble protein, denaturation-refolding was required. Results establish that the BC1-encoded MP from DNA-B is highly insoluble in solution and has a secondary structure which predominantly consists of β-strands. Its predicted large unfolded domain at the C-terminus presents some interesting speculations as to the function of the MP C-terminus. A study on Abutilon mosaic virus has revealed a homo-oligomerisation of the C-terminus of the MP and suggests that the virus possesses a host-plant species-independent capacity to bind to plasma membranes, likely occurring at the C-terminus. AbMV MP-mediated intracellular sorting is dependent on an ‘anchor domain’ (amino acids 117 to 180), which is necessary to traffic the MP complex to the cell periphery. We propose that SACMV MP has the ability to bind ATP in its native form due to ATPase activity, as demonstrated by the in vitro results herein. Viral MPs change in conformation in the presence of ATP and adopt a different conformation when denatured, compared with their native state. This suggests that, in the plant cell in vivo, native forms of the SACMV MP, or alternatively MPs that form a high-order oligomer, may undergo conformational changes when bound to biomolecules such as ATP. Indirect binding of other ligands with ATPase activity, such as HSP70, has been shown in other geminivirus studies, and recently


Hepatocellular carcinoma (HCC) is the most prevalent pathological type of liver cancer worldwide with high mortality and poor prognosis. N6-methyladenosine (m6A) can modify RNAs such as mRNA, lncRNA, miRNA, and tRNA, thereby playing a critical role in the pathogenesis of HCC. However, the role of m6A-associated small nuclear RNA (snRNA) in the prognostic value and immunotherapeutic response in HCC remains unclear.In this study, snRNA expression data, gene mutation data, and clinical data of HCC patients were acquired from The Cancer Genome Atlas (TCGA) database. We used the least absolute shrinkage and selection operator (LASSO) Cox regression analysis to identify significant prognostic m6A-associated snRNAs, and then developed a multivariate Cox model based on the selected snRNAs. HCC patients were split into low- and high-risk groups based on the median risk score. We subsequently performed Kaplan-Meier curve analysis to estimate overall survival (OS) by clinicopathological characteristics and tumor mutational burden (TMB) status in low- and high-risk HCC patients. Finally, we compared the immunotherapeutic response as represented by tumor immune dysfunction and exclusion (TIDE) scores between the two risk groups.Eight m6A-associated snRNAs were selected as independent predictors to develop the risk model. Our results revealed that the OS of HCC patients in the high-risk group was significantly worse than that in the low-risk group on clinicopathologic characteristics, including age (≤65 years and >65 years), gender , grade (G I-II and G III-IV) and TNM staging (Stage I-II and Stage III-IV). In addition, the OS of low-TMB and low-risk group was longer than that of high-TMB and high-risk group. The TIDE score indicated that HCC patients in the high-risk group were more susceptible to immunotherapy.Our study suggests that m6A-associated snRNAs may be useful biomarkers for the prognosis of HCC and that m6A-associated snRNA models can predict the effect of immunotherapy in HCC patients. Primary liver cancer (PLC) is a prevalent malignant tumor, ranking sixth in incidence and third in mortality among all types of tumors in the world . Hepatocm6A is the most important internal modification of RNA epitranscriptomes in eukaryotes . m6A-medSmall nuclear RNAs are non-coding RNAs of approximately 150 nucleotides which are commonly observed in the splicing speckles and Cajal bodies in eukaryotic cells . In the In this study, we aimed to explore the role of m6A-associated snRNAs in predicting the prognosis and immunotherapeutic response of HCC patients. We developed a risk model based on m6A-associated snRNA to analyze the OS of HCC patients on clinicopathologic characteristics  and TMB. We also used TIDE scores to analyze the effect of immunotherapy based on the m6A-associated snRNA risk model. Our study may be helpful for the guidance of personalized immunotherapy for HCC patients.2 (fold change) | > 1 and adjusted p < 0.05. Since all of these data from TCGA are public, no ethics committee approval is required.We obtained RNA-seq transcriptomic data, clinical information  Table 1,P < 0.05. Then, the R package “ggalluvial” was used to visualize the association network between 23 m6A-regulator genes and snRNAs.We obtained expression profiles of 23 m6A regulators from transcriptome data. 95 m6A-associated snRNAs were identified by the Pearson’s correlation test with the standard of Pearson cor > 0.1 and i × expri, where coefi indicated the coefficient of the corresponding m6A-associated snRNA, and expri represented the expression level of the m6A-associated snRNA. According to the median risk score, the HCC patients were split into high- and low-risk groups. Subsequently, principal component analysis (PCA) was conducted to observe the distributions of the low- and high-risk groups. The distribution of risk scores, survival status and heatmaps of m6A-associated snRNAs were evaluated in the training and testing datasets.To identify potentially optimal m6A-associated snRNAs, we randomly split HCC patients from TCGA database into training and testing datasets in a 1:1 ratio. First, nine significant prognostic m6A-associated snRNAs were filtered out by univariate Cox regression analysis. Then, Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analysis was employed to confirm the nine significant prognostic m6A-associated snRNAs with penalty parameters estimated by 1000-fold cross-validation. Finally, multivariate Cox regression analysis was conducted to establish a risk model based on eight m6A-associated snRNAs which were selected from the nine significant prognostic snRNAs in this process. Risk score was computed by the following method: Risk score =Σn i=1 coefUnivariate and multivariate Cox regression analyses were used to assess the accuracy of prognostic m6A-associated snRNA signatures as independent risk factors compared with other clinicopathological characteristics  in HCC patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the accuracy of m6A-associated snRNA models in predicting HCC prognosis, and the area under the curve (AUC) was calculated.P value less than 0.05 indicated a statistical significance.Kaplan-Meier survival analysis was performed to assess OS based on clinicopathological characteristics in low- and high-risk HCC patients by using the R package “survMiner”. A matched comparison was conducted on clinicopathological characteristics, including age (≤65 years and >65 years), gender , grade (G I-II and G III-IV), and TNM staging (Stage I-II and Stage III-IV). A P value less than 0.05 indicated a statistical significance.We applied the R package “maftools” to evaluate gene mutation data in low- and high-risk HCC patients. TMB was evaluated according to tumor-specific mutated genes. Kaplan-Meier curve analysis was conducted to assess OS in HCC patients based on TMB status. A P value less than 0.05 indicated a statistical significance.We conducted Gene Ontology (GO) functional enrichment analysis  to explore the potential signaling pathways of m6A-associated snRNAs in HCC. R package “clusterProfiler” was utilized to perform this analysis. Subsequently, significant GO terms were visualized in a bar graph by using “enrichplot” package. In addition, we explored the immune functions in HCC patients based on m6A-associated snRNA model, and distinguished the difference in immune activities and functions between low- and high-risk HCC patients. This process utilized the R package “pheatmap”. A P value less than 0.05 indicated a statistical significance.Tumor Immune Dysfunction and Exclusion (TIDE) is a computational framework for assessing the potential of tumor immune escape . A low TThe flow diagram of this study was shown in We conducted LASSO-Cox regression analysis to identify nine significant prognostic snRNAs selected from 95 m6A-associated snRNAs to construct risk scores for the prediction of prognosis in HCC patients  were 1.239 and 1.134–1.354 (p < 0.001), respectively , gender , grade (G I-II and G III-IV), and TNM staging (Stage I-II and Stage III-IV). However, female high-risk HCC patients did not have significantly poorer OS than those low-risk HCC patients , thus alleviating HSC activation and suppressing hepatic fibrosis and carcinogenesis . Iwai etOur study suggested that m6A-associated snRNAs may be useful biomarkers for the prognosis of HCC and that the m6A-associated snRNA model could predict the effect of immunotherapy in HCC patients. Our study provided new clues to the potential biological mechanism of m6A-associated snRNAs in HCC.The original contributions presented in the study are included in the article/CZ, WZ, SD, and QW conceived and designed the research. CZ, YS, PL, and ZT analyzed the data. All authors made substantial contributions towards drafting the manuscript, reviewed the final manuscript for intellectual content, and authorized the submission. All authors contributed to the article and approved the submitted version.This work was supported by the grants from the National Natural Science Foundation of China (82073332), the Natural Science Foundation of Zhejiang Province (LQ19H030002), the Medical Science and Technology Project of Zhejiang Province (2023KY182), and the Youth Fund of the Affiliated Hospital of Hangzhou Normal University (2021YN2021027).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here, we investigated the applicability of the formula (K-index) to predict the response to treatment with another ICI to expand its clinical applicability. Furthermore, we developed a simpler assay method based on whole blood (WB) samples to overcome the limitations of the PBMC method, such as technical difficulties, in obtaining the K-index.Biomarkers that can accurately predict the efficacy of immune checkpoint inhibitors (ICIs) against programmed death 1 (PD-1) ligand in cancer immunotherapy are urgently needed. We have previously reported a novel formula that predicts the response to treatment with second-line nivolumab with high sensitivity and specificity in patients with non-small cell lung cancer (NSCLC) previously treated with chemotherapy. The formula was based on the percentages of CD62LThe K-index was evaluated using the PBMC method in 59 patients with NSCLC who received first-line pembrolizumab treatment. We also assessed the K-index using the WB method and estimated the correlation between the measurements obtained using both methods in 76 patients with lung cancer.This formula consistently predicted the response to first-line pembrolizumab therapy in patients with NSCLC. The WB method correlated well with the PBMC method to obtain %Teff, %Treg, and the formula value. The WB method showed high repeatability . The data obtained using WB samples collected in tubes containing either heparin or EDTA-2K and stored at room temperature (18–24 °C) for one day after blood sampling did not differ. Additionally, the performance of the WB method was consistent in different flow cytometry instruments.The K-index successfully predicted the response to first-line therapy with pembrolizumab, as reported earlier for the second-line therapy with nivolumab in patients with NSCLC. The WB method established in this study can replace the cumbersome PBMC method in obtaining the K-index. Overall, this study suggests that the K-index can predict the response to anti-PD-1 therapy in various cancers, including NSCLC.The online version contains supplementary material available at 10.1186/s12885-022-10445-2. Immune checkpoint inhibitors (ICIs) targeting the anti-programmed death 1 (PD-1) ligand have shown epoch-making effects in several cancers, especially in terms of long-term survival . However+ T cell immunity in the peripheral blood before therapy predicted the antitumor efficacy of second-line nivolumab therapy in patients with non-small cell lung cancer (NSCLC). Furthermore, we developed a formula to predict non-responders to nivolumab based on the percentage of CD62LlowCD4+ and CD4+CD25+FOXP3+ T cells in CD4+ T cells of the peripheral blood, which demonstrated a sensitivity and specificity of 85.7 and 100%, respectively ). The mPFS was 363.0 days in the group with an index ≥192 and 122.5 days in the group with an index < 192  of 206 days and a 12-month PFS of 37.1%, which was similar to the subgroup analysis of KEYNOTE-042 , which rAs the K-index was useful for predicting the response to pembrolizumab, and its significance was confirmed, we aimed to develop an easier method to measure the index. An outline of the PBMC and WB methods is shown in Fig. + T cells were gated using a CD4+/SSC-A dot plot  obtained by excluding dead cells from PBMCs using CyTOF  were corThe anticoagulants Heparin and EDTA-2K are commonly used during blood collection for blood cell analysis . We inveDepending on the circumstances, the collected blood samples were measured either immediately or at another laboratory. Therefore, we investigated whether blood samples could be stored for measurement using the WB method. Blood samples from patients with lung cancer were stored in heparin-containing blood collection tubes at room temperature (18–24 °C) for 1 d after blood sampling. The values of %Teff, %Treg, and the K-index of the same sample measured within 4 h after blood collection and after storage for 1 d Fig. a–c were Finally, we examined whether the WB method can be applied to different FCM instruments. The same blood samples from patients with lung cancer prepared using the WB method were measured simultaneously using a BD FACSCanto II and Sysmex XF-1600. The measured results were well correlated in both measurements using FACSCanto II and XF-1600 Fig. a–c, indiVarious PD-1 inhibitor combination therapies are being developed to enhance T cell restoration and increase the probability of long-term survival of patients with cancer –23. Howe+ T cells  (X-axis) and this report (Y-axis). (a–c) Twenty commercially available PBMC  were stained with CD4-BV650 , CD25-PE-CF594 , CD62L-BV421  and anti-FOXP3-PE  as the antibody panel in the previous report. The samples were analyzed using a BD FACSLyric™ (BD Biosciences). The correlation of the data obtained by both panels is shown as %Teff (a), %Treg (b), and the K-index (c). Regression lines and correlation coefficients were calculated for %Teff, %Treg, and the K-index. The gray line represents the regression line, the light gray line represents the 95% confidence interval, and the dotted line represents the 95% prediction interval. PBMC, peripheral blood mononuclear cells; Teff, effector T cells; Tregs, regulatory T cells.Additional file 2: Supplementary Fig. 2. Overall analysis data of response to initial pembrolizumab therapy in patients with NSCLC. Analysis of mPFS (a) and mOS (b). mOS, median overall survival; mPFS, median progression-free survival; NSCLC, non-small cell lung cancer; PD, progressive disease; PR, partial response; SD, stable disease.Additional file 3: Supplementary Fig. 3. Correlation between measurements obtained using the PBMC and WB methods. The measurements were performed using CyTOF in the PBMC method and FCM in the WB method. The gating strategy of CyTOF analysis was performed as described in a previous study [18]. In brief, dead cells were removed using 198Pt staining. The analysis of the lower layer of CD4+ cells was the same as the FCM analysis. Blood samples were collected from 53 patients with lung cancer. (a–c) Each sample was divided into two and measured using PBMC and WB methods. Regression lines and correlation coefficients were calculated for %Teff (a), %Treg (b), and the K-index (c). The gray line represents the regression line, the light gray line represents the 95% confidence interval, and the dotted line represents the 95% prediction interval. PBMC, peripheral blood mononuclear cells; Teff, effector T cells; Tregs, regulatory T cells.


Goniothalamus umbrosus after purification with petroleum ether. The isolated SPD was then subjected to gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses for structure validation. The cytotoxicity of the SPD was evaluated using a cell viability assay, while the anti-viral activity of the SPD towards DENV-2 was confirmed by conducting a foci reduction assay which involved virus yield reduction, time-of-addition, and time removal assays. Transcriptomic analysis via quantitative real-time polymerase chain reaction (qRT-PCR) using the DENV-2 E gene was conducted to investigate the level of gene transcript. Immunocytochemistry analysis was used to investigate the effects of SPD treatment on protein E expression. Finally, software molecular docking of the SPD and E protein was also performed. The cytotoxicity assay confirmed that the SPD was not toxic to Vero cells, even at the highest concentration tested. In the time-of-addition assay, more than 80% foci reduction was observed when SPDs were administered at 2 h post-infection (hpi), and the reduction percentage then dropped with the delay of the treatment time, suggesting the inhibition of the early replication cycle. However, the time removal assay showed that more than 80% reduction could only be observed after 96 h post-treatment with the SPD. Treatment with the SPD reduced the progeny infectivity when treated for 24 h and was dose-dependent. The result showed that transcript level of the E gene in infected cells treated with the SPD was reduced compared to infected cells without treatment. In immunocytochemistry analysis, the DENV-2 E protein exhibited similar expression trends, shown by the gene transcription level. Molecular docking showed that the SPD can interact with E protein through hydrogen bonds and other interactions. Overall, this study showed that SPDs have the potential to be anti-DENV-2 via a reduction in viral progeny infectivity and a reduction in the expression of the DENV-2 E gene and protein at different phases of viral replication. SPDs should be further researched to be developed into an effective anti-viral treatment, particularly for early-phase dengue viral infection.A study was conducted to investigate the anti-viral effect of a styrylpyrone derivative (SPD) called goniothalamin and the effects on the dengue virus serotype 2 (DENV-2) replication cycle. The SPD was prepared from the root of  Aedes aegypti mosquito. Dengue virus infection may be asymptomatic or may lead to undifferentiated fever, dengue fever (DF), or dengue hemorrhagic fever (DHF) with plasma leakage which may lead to hypovolemic shock  [Dengue infection is caused by the dengue virus (DENV) which belongs to the genus Flavivirus in the family Flaviviridae. There are four serotypes of dengue virus: DENV-1, DENV-2, DENV-3, and DENV-4 [Carica papaya or papaya has been used in traditional medicine as an alternative to treat dengue infection in humans [C. papaya leaves was found to have an effect in increasing platelet count in study mice infected with DENV-2 [C. papaya leaf extract can have a healing effect by compensating for mineral deficiency caused by DENV infection and strengthening the immune system against DENV infection [Euphorbia hirta is another popular plant that is used in traditional medicine to treat dengue fever by people in the countryside areas of the Philippines [At present, no natural ingredients have been certified to treat dengue infection, although many traditional medicinal plants have been reported to have strong anti-viral activity, and some of them have already been used to treat animals and people who suffer from viral infection by inhibiting the replication of several viruses ,5,6. Forn humans . Infectin humans ,9. In vih DENV-2 . In addinfection ,12. Euphlippines . A commelippines . Goniothalamus umbrosus, locally known as `kenerak’ in Malaysia, is a tropical plant under the Annonaceae family which is well-distributed across Peninsular Malaysia. This plant has long been used traditionally as contraceptive, anti-pyretic, anti-malaria, and as post-partum treatment [Goniothalamus sp. have been studied quite extensively, resulting in the isolation of alkaloids, steroids, terpenoids, and cardiac glycosides from bark and leaf extracts of this plant. reatment ,15. WatePhytochemical compounds have been recognized to have medicinal significances. For example, many alkaloids derived from medicinal plants show biological activities such as anti-viral, anti-bacterial, anti-inflammatory, and anti-cancer properties . SteroidGoniothalamus sp. and found a total of 110 compounds based on gas chromatography–mass spectrometry (GC-MS) detection [Goniothalamus sp. is styrylpyrone derivatives (SPDs), one of which is goniothalamin. Styrylpyrone derivatives are among styryl lactones that have been isolated from several Goniothalamus sp. [A study investigated the chemical components in hexane and ethyl acetate extracts of etection . These camus sp. . SPDs aramus sp. . This coamus sp.  and alsoamus sp. . Figure amus sp. . This phamus sp. ,32, an aamus sp. .The identification of a pure compound can be determined using gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR). NMR spectroscopy is an analytical chemistry technique used to determine the structure, dynamics, reaction state, content, and purity of an organic compound . GC-MS iG. umbrosus was observed to be safe and non-toxic to cells, protect cells against viral infection, inhibit viral attachment and penetration, and also have a virucidal effect [We have previously reported that SPD isolated from l effect . This stG. umbrosus after purification with petroleum ether. The characteristics of an ununiformed clear crystal with pointed/sharp ends were observed. Tiny, needle, white and solid crystalline structure of SPD was obtained after several recrystallization using petroleum ether. This crystal was then subjected to GC-MS and NMR analysis for structure validation. The purity of the isolated SPD was validated using GC-MS. The GC-MS spectrum showed one peak with a mass to charge ratio (m/z) of 200 which was consistent with the SPD’s molecular weight [Pure SPD (0.05 g) was isolated from r weight . Our resr weight . Major f1H NMR spectra for the isolated SPD in this study showed the signal at δ 7.0 for H-6 and δ 6.0 for H-3 as olefinic protons of lactone skeleton. Two more olefinic protons appeared at δ 6.4 and δ 6.7 which had H-4 and H-5 protons, respectively. The signal that appeared at δ 5.1 was an H-2 proton. The signal at δ 7.3 was a proton of the phenyl group with an H-8 proton. The structure of the SPD determined in this study was similar to the structure of goniothalamin , as shown in The cytotoxic concentration, CCThe anti-viral study was conducted using focus reduction assay. The calculation of focus inhibition is the basis of calculating the percentage of virus reduction. Time-dependent studies were conducted to evaluate the effect of delayed treatment and also the effect of different time exposures to the SPD against its DENV-2 anti-viral activity. In the viral yield reduction assay, a concentration-dependent reduction in virus titer percentage was observed after 48 h post-treatment with SPD. The calculated virus titer revealed significant differences between all SPD concentrations. The treatment with the highest concentration (15 μM) resulted in the lowest titer produced, as shown in In the time-of-addition study, SPD treatment was observed to be most effective when added at two hours post-infection (hpi) . More thA time removal assay was carried out to evaluate the minimum time of SPD treatment that conferred the optimum anti-DENV-2 activity. Treatment with the SPD began at 2 hpi and the removal of the SPD continued for every three hours until 15 hpi. Low plaque reduction was observed as early as 6 hpi (four hours post-treatment) . As earlp value = 0.4392. However, the decrease in E gene expression was significant (p < 0.05) at 6 hpi. It is likely that the SPD needed time to be absorbed and act on the E gene.Quantitative RT-PCR was employed to monitor the transcription levels of the DENV-2 E gene within 120 h of replication in Vero cells. The effect of the SPD treatment at the concentration of 12.5 µM on DENV-2 gene expression was evaluated from time interval samples of 2, 6, 12, 24, 48, 72, 96, and 120 hpi. The E gene was selected as the target gene in this study to represent the early phase in the DENV-2 replication cycle. The E gene is required in the process of attachment and entry of the virus into the host cell ,41. The Immunofluorescent staining analysis was performed to confirm the results of quantitative RT-PCR analysis and, at the same time, to prove the effect of the SPD on the attachment process involving the DENV-2 E protein in infected and treated cells. The molecular docking of the SPD was carried out on the DENV-2 E protein. SPD was shown to bind to domain III of the DENV-2 E protein, spanning from amino acid residues 274 to 275, at a binding affinity of −1.86 Kcal/mol. The binding pose of the SPD on DENV-2 E protein is shown in andersonii, G. macrophyllus Miq., and G. malayanus [G. cardiopetalus Hook.f. & Thoms [G. amuyon [G. griffithii [Goniothalamus sp. This was proven when 5-isogoniothalamin oxide was successfully isolated from the stem bark of G. sesquipedalis [G. umbrosus against DENV-2.Goniothalamin was first reported as an active constituent of the bark of G. alayanus . Since t & Thoms , aerial . amuyon , and rooiffithii . Petroleipedalis . Here, wG. umbrosus as goniothalamin (SPD) based on a previous study [m/z value of 200, which confirmed that the isolated compound was pure. The stability evaluation of the SPD was conducted based on the physical appearance, pH, and drug content according to a previous study [Pure compounds can be isolated from crude extract. Crude extract is usually prepared via several means, namely maceration, decoction, digestion, infusion, percolation, and hot continuous extraction (using Soxhlet apparatus). In this current study, we used hot continuous extraction using Soxhlet apparatus because this technique poses advantages due to its simple approach, low cost, and efficient extraction owing to the constant displacement of fresh solvent which comes in contact with plant material. The further isolation of pure SPD from crude extract was achieved by using simple crystallization from crude extract . Petroleus study  with a cus study . No obvius study .50). The cytotoxicity analysis via the crystal violet dye assay was made concurrently with the anti-viral activity assessment to ensure that the potential virus-specific compound had low or no effect on cellular metabolism and thus showed no toxicity effect against the host cell [50 value, lower concentrations which were least toxic or non-toxic to the cells were used in anti-viral screening. In this study, a 3--2,5-diphenyltetrazolium bromide (MTT) assay was utilized in the cell viability assessment by measuring the ability of mitochondria to reduce tetrazolium salt to insoluble formazan crystal [50 was considered to be not in the range of cytotoxic compounds since the value of CC50 was determined to be higher than the minimum value for a compound with active cytotoxicity. Any CC50 or IC50 value of a compound that was less than 4 μg/mL was considered to have an active cytotoxic effect [A cytotoxicity assay was performed ahead of anti-viral screening to determine the SPD concentration that was cytotoxic to 50% of the cell population  and was further purified with petroleum ether to yield a crystal compound of the SPD. The purity of the compound was confirmed via FT-NMR 400 MHz (Bruker Avance 111) and gas chromatography (QP2010 Ultra) using the SPD from [5-hydroxy-7-phenylhepta-2,6-dienoic acid-δ-lactone (SPD) was isolated from SPD from  as the s4 cells per well in 96-well, flat-bottomed microtiter plates  supplemented with 5% fetal bovine serum (FBS) and incubated at 37 °C overnight. Upon confluency, growth medium was removed and 100 μL of SPD from several concentrations (3–100 µM) was added. Cells were further incubated at 37 °C in a 5% CO2 atmosphere for 48 h. Cells without compound treatment served as base controls. After 48 h of incubation, the cells were washed gently, stained with MTT (5 mg/mL) for a minimum of two hours, and lysed with dimethyl sulfoxide (DMSO) to dissolve the formazan crystals. Color development was detected using a microplate reader (TECAN Infinite 200 PRO) at 540 nm. The percentage of living cells was calculated via comparison with healthy untreated cells. A dose–response curve was plotted using Graph Pad Prism 5, and the half maximal cytotoxic concentration (CC50) of the SPD was determined from the plot. The concentration of the SPD which showed at least 80% cells’ viability in the cytotoxicity assay was chosen as the starting concentration used throughout the experiments. This concentration was important for the anti-viral assays in the next step. The assay was performed as described in a published study . The cytThe anti-viral evaluation of the SPD towards DENV-2 was conducted using a standard foci reduction assay based on a published study  with sliThe assay was conducted according to a published study  with modw/v). The number of DENV-2 foci was counted using a stereomicroscope, and the titer of virus was expressed as foci forming units (FFUs) [Antiviral activity was determined by measuring the reduction in the number of viral foci by following a previously described assay . Monolays (FFUs) . The perThe assay was conducted according to a previous study  with modThis assay was conducted according to a published procedure , with mihttp://www.chemspider.com) (accessed on 25 July 2019).The docking of the SPD to the DENV-2 E protein was conducted using the program AutoDock Vina . The thrThe assay was conducted according to a published procedure  with slip value < 0.05.Statistical analysis was performed using Graph Pad Prism for Windows, version 5 . Data were analyzed using the Student’s t-test. All analyses used a 95% CI , and the difference obtained was significant if the In this study, the SPD was confirmed to have anti-DENV-2 activity when administered to DENV-2-infected Vero cells by inhibiting the mechanism of viral attachment. SPD is proposed to inhibit the E protein during the early stages of the virus life cycle and hence suppresses early viral gene transcription, replication, and translation. An in vivo experiment is recommended to be performed to determine the efficacy of the SPD for its therapeutic ability as an anti-dengue agent.


Objective: To evaluate SARS-CoV-2 genome detection using pooled samples by RT-qPCR assay, compared to individual samples. Method: At first all samples were tested individually using two commercial methods targeting ORF1ab, NP and E genes. Then, four experimental groups of samples were pooled and evaluated using the same detection methods. Findings: Compared to the individual sample testing, the sample pooling conserved the sensitivity of the detection in all groups of pooled samples when the Ct value in single test was lower than 33. Conclusion: Specimen pooling may fail to detect positive samples with high Ct values. However, in scarcity of reagents or in population surveys, it could be considered as an alternative method in low prevalence settings. Since SARS-CoV-2's first emergence in Wuhan (China), late December 2019, it caused a large pandemic . As of MThe variable incubation period of COVID-19 , the rolThe throat swabs in viral transport medium were sent routinely via Ministry of Health and Education to the Iran National Influenza Center (NIC) located at Tehran University of Medical Sciences, for SARS-CoV-2 testing under cold chain transport. A one-step real-time RT-PCR for SARS-CoV-2 has been routinely performed on all specimens. To evaluate the usefulness of pooling samples of SARS-CoV-2 genome detection by real-time PCR, from previously tested specimens for routine diagnosis, 15 positive specimens with CT value of 16–40 and 29 negative samples were randomly chosen. Then, 60 experimental pools were created using 200 microliters of one SARS-CoV-2-positive throat specimen mixed with four batch sizes comprising of 4, 9, 19 and 29 negative samples . The second method involved targeting primers and probes for E gene  using Invitrogen master mix and enzyme (SuperScript III Platinum One-Step qRT-PCR Kit). The assays were conducted on a StepOnePlus ™ Real-Time PCR Systems  following the manufacturer's instructions. In the next step, E gene-positive samples were tested using the RpRd gene  recommended by WHO. The cutoff PCR Ct of both methods was ≤40.For both individual and pooled samples, 200 μl of specimens were subjected to viral RNA extraction using the High Pure Viral Nucleic Acid Kit , according to the manufacturer's instruction. Extractions were stored at -80°C until the next steps. A one-step real-time RT-PCR was performed using two methods. The first method targeted the ORF1ab gene, SARS-CoV-2 was detected providing non-pooled positive samples displaying RT-PCR Ct <33 in all groups of 5, 10, 20 and 30 samples. The negative pooled samples did not have any CT and were not seen in the figure. NP-positive samples were detected as long non-pooled positive samples displaying RT-PCR Ct ≤35 in groups of 5, 10 and 20 samples and Ct ≤34 in the group of 30  recommended by WHO. The result is shown in The samples that tested positive for the Orf1ab real-time RT-PCR for the original individual specimen was <33 in pooled samples, one positive clinical sample could be detected. Positive samples with a low viral load  in pooled samples were not detected . That is, provided the Ct value of detected . It shouRpRd gene in E gene in E gene giving lower Ct values compared to the ORF1ab-targeting primers. Herein, Ct variations between original samples and pooled samples were in the range of 2.0 to 4.0 units in the group of 5 samples and 3.0 to 5.0 in the group of 10 samples. It was hypothesized that, if the Ct difference is less than 3 in any of the studied batch size, a positive sample with the Ct up to 35 might be detectable when pooled with negative samples. Also, if the Ct difference is greater than 3, the likelihood of a false-negative result would increase. In this study, a range of 5 to 30 samples per pool showed similar real-time RT-PCR results, consistent with previous reports performed on the similar sample sizes per pool [This study was the first research using pooled samples for COVID-19 diagnosis with common and available real-time PCR methods in Iran. It was performed during May 2021 when the infection prevalence was 0.15%. The results showed both methods preserved the sensitivity of the tests to detect a positive sample except when the Ct value of the non-pooled positive sample was higher than 33 –2. This per pool ,14,23. Tper pool ,25. It hper pool . Pooled per pool ,12. To fper pool ,27. Thisper pool . The higper pool . HoweverCollectively, our results depict that specimen pooling using common and available RT-PCR testing in Iran and most of the countries in Middle East, might result in missing persons with a mild infection. When there is a shortage of resources, this could be a challenge for persons with a high vulnerability to acute COVID-19 infection or hospitalized patients.Since world experiences a shortage of diagnostic kits for SARS-CoV-2, pooling of several specimens was specially performed in asymptomatic carriers with low infection rates in the screening programs.The detection of a positive clinical sample was independent of the pool size up to 30 samples.Orf1ab real-time RT-PCR for the original individual specimen was <33 in pooled samples, one positive clinical sample could be detected.Provided the Ct value of Specimen pooling using the alternative time RT-PCR testing might result in missing persons with a mild infection that this could be a challenge for persons with a high vulnerability to acute COVID-19 infection or hospitalized patients.


In oral administration systems, mucoadhesive polymers are crucial for drug localization and target-specific activities. The current work focuses on the application of thiolated xanthan gum (TXG) to develop and characterize a novel mucoadhesive nanocrystal (NC) system of simvastatin (SIM). Preparation of SIM-NC was optimized using response surface methodology (RSM) coupled with statistical applications. The concentration of Pluronic F-127 and vacuum pressure were optimized by central composite design. Based on this desirable approach, the prerequisites of the optimum formulation can be achieved by a formulation having 92.568 mg of F-127 and 77.85 mbar vacuum pressure to result in EE of 88.8747% and PS of 0.137.835 nm. An optimized formulation was prepared with the above conditions along with xanthan gum (XG) and TXG and various parameters were evaluated. A formulation containing TXG showed 98.25% of SIM at the end of 96 h. Regarding the mucoadhesion potential evaluated by measuring zeta potential, TXG-SIM-NC shoed the maximum zeta potential of 16,455.8 ± 869 mV at the end of 6 h. The cell viability percentage of TXG-SIM-NC (52.54 ± 3.4% with concentration of 50 µg/mL) was less than the plain SIM, with XG-SIM-NC showing the highest cytotoxicity on HSC-3 cells. In vivo pharmacokinetic studies confirm the enhanced bioavailability of formulated mucoadhesive systems of SIM-NC, with TXG-SIM-NC exhibiting the maximum. Nanomedicines have opened up new opportunities for therapeutic treatments because biocompatible nanosize-based particles may enhance the overall pharmacological properties of a given drug substance, including improved bioavailability  and targGlobally, there were 19.3 million new cancer diagnoses and 10.0 million cancer deaths in 2020, estimated by the Global Cancer Observatory (GLOBOCAN). In addition to being the leading cause of death worldwide, cancer also makes it challenging to increase life expectancy across the board . ChemothDue to the weak and heterogeneous enhanced permeability and retention (EPR) effect in solid tumors in the context of human tumors, the use of commercial nanomedicines for chemotherapy based on passive targeting, such as drug-loaded liposomes, micelles, and nanoparticles, has encountered a bottleneck . Drug deNatural gums and mucilage of plants are impervious as a pharmaceutical excipient, particularly in the formulation of controlled drug forms . These sThiolation of the mucoadhesive polymers provides the potential to make disulfide bonds (inter-/intrachain) within the polymeric system and can significantly enhance their cohesive nature. The chemical reaction of thiol moiety with mucin-containing cysteine results in the development of strong covalent bonds ,21. ThioSimvastatin (SIM) is significantly metabolized by microsomal enzymes and has a lower bioavailability (5%). SIM is a biopharmaceutics chemical that falls under the Biopharmaceutics Classification System (BCS) Class-II category. It has low aqueous solubility and a good permeability through biomembranes. The cytochrome enzyme CYP3A4 primarily targets the lactone structure of SIM and significantly reduces intestinal absorption. Low bioavailability is caused by the medicine’s aquaphobic character, which prevents complete drug dissolution in the intestinal medium. Statins are generally effective in maintaining cholesterol levels and lower blood cholesterol by inhibiting 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) ,23. SIM The response surface methodology (RSM) is a group of statistical methods used to create and assess the relationship between a response and a set of relevant variables. It is possible to study the optimization process using the data gathered in this way from an experiment . RSM is Biocon Pvt Ltd., Bangalore, India generously provided SIM. Pluronic F-127, chloroform, dextrose and xanthan gum were procured from Loba Chemie Pvt Ltd., Mumbai, India. All other chemicals used were of analytical grade.The NCs were prepared using a three-phase nanoparticle engineering technique (3PNET), which contains phases 1 and 2 of hydrated amorphous aggregate, phase 3 of stabilized NCs, and phase 1 of amorphous precipitate . In a nu1) and vacuum pressure (X2) were selected as independent parameters at five different values and are coded as −1.414 (low), −1, 0 (medium), +1, and +1 (high) [The statistical model RSM standardized the synthesis of SIM-NC. The F-127 (X1 (high) ,32,33  analyzed the SIM-NC average PS, PDI, and electrokinetic potential. To prevent the blockage of particles, the prescribed amount of SIM-NC was re-dispersed into a generous amount of Milli-Q water and vortexed for 5 min. At 25 °C, the final sample was evaluated in triplicate for 1 min .g for 20 min to test SIM encapsulation [The NCs were kept at room temperature for 1 h, passed through 0.45 and 0.22 mm centrifugal filters and a 3000 MWCO microcon , and centrifuged at 16,000× sulation . A waterSoftware called Design-Expert was used to trigger the reactions that all of the preparations had to provide. The study technique and the response surface graph were developed using the responses. A numerical standardization method created an optimal formula with the lowest and maximal limits for each parameter. A desirability function was created by incorporating the results. The answers that satisfied the criteria were documented, and the set of options was ranked in order of desirability. The response surface graph clarified the relationship between the independent and dependent parameters. ANOVA was used to examine how different variables affected the slope coefficients . The difA scanning electron microscope (SEM)  was used to examine NCs’ morphology. Raw ETO MC powder and processed NC powder were applied to a double-sided tape, coated with a 30 nm coating of gold, and then subjected to a 2 min period of vacuum (10-6 Pa) and SEM observations at a 15 kV accelerating voltage .An analytical method known as XRD is quick and is generally used to determine the phase of crystalline materials. Crystallinity of pure SIM and SIM-NC formulations was investigated utilizing XRD analysis using Inxitu Benchtop XRD .To examine release kinetics, 10,000 Da dialysis cassettes were filled with 1 mL of O-SIM-NC and submerged in 400 mL of dialysis buffer (PBS (pH 7.4) containing 0.05% Tween 80). At particular times, 2 mL aliquots of buffer were UV spectroscopically measured . To mainThe mucoadhesive assessment of both SIM-NC formulations was evaluated in vitro using two different techniques ,40. In tUsing an in vitro model and the MTT assay, the cytotoxicity of the standardized formulation (O-SIM-CAN) was assessed in HCS-3 cells . HCS-3 cells were trypsinized using 0.25% trypsin-EDTA and put in 96-well plates (about 2500–5000 cells/well) at a density of 5 104 cells/mL. The entire medium was then replaced with Dulbecco’s modified Eagle medium (DMEM)  after 24 h. The absolute test samples were given to the other cells in the range of 10–50 g/mL, whereas 5-fluorouracil was administered to the reference cells. After 72 h of incubation, 0.1 mL of DMEM containing 0.2 mg/mL MTT was added, and the mixture was incubated for an additional 2–3 h. DMSO was used in place of DMEM to eliminate the developed formazan. A microplate reader  was used later to measure the absorbance at 540 nm. IC 50 values were calculated when the dose–response relationship was established .In vivo studies were conducted after obtaining approval from the Animal Ethical Committee of the Institution of the Clinical Laboratory Center, Beni Suef, Egypt . PK solver software was used to analyze the pharmacokinetic (PK) performance of NC after oral and transdermal delivery. Wistar albino adult male rats weighing approximately 180–250 g were used. The following is a breakdown of a single dose trial of four groups of six animals each. Simvastatin suspension (10 mg/kg) was used in Test Group I. XG-SIM-NC (10 mg/kg) and XG-SIM-NC (10 mg/kg) were used in Test Groups II and III.Prior to receiving medication formulations, animals fasted for 24 h while still having unrestricted access to water. The abdominal hair was cut the day before the experiment by using a depilatory product for 10 min and then washing it with distilled water. The animals were sedated with ketamine  and placed in a supine position on the day of the experiment. With the aid of an oral feeding needle, all test samples were given orally (10 mg/kg). A retro-orbital puncture was used to collect blood samples totaling roughly 0.5 mL at intervals of 0.5, 2, 4, 8, 12, 24, and 48 h following oral administration. Capillary tubes were used to transfer the samples from a retro-orbital puncture into a glass tube that had been heparinized and included the anticoagulant ammonium oxalate (1% solution). The plasma was promptly separated by microcentrifugation at 5000 rpm and then kept at 20 °C pending HPLC analysis ,44.After centrifuging 1.5 mL of animal blood at 5000 rpm for 5 min, 0.75 mL of plasma was recovered. Then, 0.5% trichloroacetic acid was added to this sample. Medicine was separated from plasma at 4000 rpm for 15 min at 4 °C ,46. The The PK solver application shows HPLC data on time versus plasma drug concentration. Peak plasma concentration (Cmax), time required to attain Cmax (tmax), area under the curve (AUC0-t), and (AUC0-) were directly read from the individual plasma drug concentration versus time profile. Biological half-life (t1/2) and mean residence time (MRT) were also calculated using PK solver software .The effect of particular variables and how they interacted to produce the minimal PS and greatest EE were studied using CCD. Thirteen experimental trials in total were anticipated, and F-value, p-value, and R2 values were considered. The largest polynomial order is seen in the quadratic model, with a p-value (degree of significance) of 0.0001  and fit summary were used to pick the quadratic model for all responses. When choosing the models, f 0.0001 .R2 of 0.8618 and the adjusted R2 of 0.9603 is less than 0.2. Adequate precision measures the ratio of signal to noise. A ratio of at least 4 is preferred. A strong signal is indicated by the ratio of 51.052. To move around the design space, this model was utilized. Similar outcomes were seen for PS  [For EE, the difference between the predicted 18.7863) .p > 0.05), which supports the fitness of the chosen design. The p-values for both responses were determined to be 0.0745 and 0.1115, respectively, indicating a non-significant lack of fit.The accuracy of all these selected models was further confirmed by the normal plot of residuals . For thiF-values of 214.68, 90.00, and 158.71. [An ANOVA was used to investigate the quantitative impacts of particular factors on responses ,50. Mult 158.71. .2, and B2 are significant model terms in the case of EE. According to the experimental plan, EE might be impacted by two factors: (i) an antagonistic effect of component B; (ii) a synergistic effect of A and AB, with AB impacts being the more significant; and (iii) polynomial terms of A and B. According to the experimental plan, PS could be impacted by the antagonistic effects of factors A and B as well as the synergistic effects of polynomial term B, with B effects having the most impact. The polynomial term of factor B and its p-value are 0.0001 and 0.0132, respectively  are essential for illuminating the interaction and primary effect .By using the desirability function (D), it was possible to optimize various models acquired through the experimental study. To produce the overlay graph, several constraints, including particle size, zeta potential, and PDI minimum, were specified for each response ,30. The The size and shape of O-SIM-NC were determined by SEM surface morphology characterization. With sizes ranging from 120 to 150 nm, naked SIM nanocrystals displayed a rod-like structure a and kepTo ascertain whether SIM would be released from nanocrystals prior to cellular absorption as a function of time, the in vitro drug release profile of SIM from XG-SIM-NC and TXG-SIM-NC was examined using two dissolution media of pH 2.0 and 7.4. Nanocrystals exhibit a significantly sustained release profile, as shown in The interaction between TXG-SIM-NC, XG-SIM-NC, and mucin was shown using two in vitro techniques. Zeta potential measurements of their dispersions were performed to gain insight into the mechanism of SIM-NC–mucin interaction. The results are displayed in The absorbance of a 0.1% aqueous mucin dispersion at 650 nm was utilized as a reference for the turbidimetric investigation. The findings of the turbidimetric experiment are displayed in Following 72 h of treatment, the cytotoxicity of the improved formulations of SIM and plain SIM against HCS-3 cells was evaluated using the MTT test. A calibration curve was created using various concentrations of simvastatin in order to determine the unknown plasma drug concentration. Simvastatin’s nominal concentration and peak area were plotted to test the calibration curve’s linearity. Simvastatin was examined at eight different concentrations  for the linearity experiments. Over the concentration range under study, the peak area response was linear. The correlation coefficient, or “r2,” was discovered to be 0.999. After a single dosage of the test, samples were administered, an unknown plasma drug concentration was obtained, and the pharmacokinetic data were effectively determined using the HPLC interpolation methodology. A calibration curve was used to determine the unknown concentration. Simvastatin’s mean plasma concentration as a function of time has been displayed, and 0-α) was determined to be 84.6528 µg/mL/h, 1453.0478 µg/mL/h, and 1847.0654 µg/mL/h, respectively. It was discovered that the mean residence time (MRT) for the thiol formulation was more significant at 98 h, which is attributable to the improved mucoadhesive capability of XG. According to the in vivo pharmacokinetics data, SIM-NC formulations showed higher AUC0-α, Tmax, and MRT with lower Cmax values when compared to plain SIM. It has been determined that XG-SIM-NC and TXG-SIM-NC exhibit an increase in bioavailability of approximately 17.16% and 21.82% compared to the SIM suspension formulation as a benchmark. Finally, the pharmacokinetic profile can be correlated with in vitro drug release pattern. The in vitro drug release profile shows maximum drug release by the end of 36 h followed by a very slow phase of drug release. By the end of 24 h, more than 70% of the drug has been released. SIM absorption in in vivo conditions can be summarized as follows: Cmax was reached at the end of 14 h, but the drug concentration was maintained within the therapeutic range till the end of 96 h. Till the end of 72 h, the pattern of SIM release and absorption was found to be similar, the slight changes in the profiles can be attributed the presence of mucus later during in vivo studies.When compared to XG-SIM-NC, it was found that TXG-SIM-NC controlled the release as well as pharmacokinetic characteristics, as shown in We reported here that a simple three-step methodology could formulate NCs of SIM. In developing the SIM-NCs, RSM, in association with various statistical estimations, has been applied to optimize the various process variables in the formulation. Based on this desirability approach, a formulation containing 92.568 mg of F-127 and 77.85 mbar vacuum pressure can accomplish the prerequisites of the optimized formulation. They resulted in EE of 88.8747% and PS of 0.137.83. Final NCs were made into suspensions using XG and TXG. The formulation made with TXG shows its excellent properties in terms of drug release and mucoadhesion potential. The mucoadhesion property further enhanced the cytotoxic nature of the formulated SIM-NC. The relative bioavailability of TXG-SIM-NC was about 21.82%, in contrast with the plain SIM suspension. Furthermore, the optimized method is believed to be suitable for formulating other hydrophobic drugs. Lastly, the NCs can be further modified for targeted delivery of anticancer drugs to increase their therapeutic efficacy.